Growth rate, meat quality and other traits are the main goals for improving fish varieties, and reproductive and developmental characteristics of fish have a very close relationship with these important traits. The Fish Energy Biology Research indicates that once the reproductive development of fish is controlled, their uptake of food will be more efficiently converted to the development of somatic cells, thus promotes growth. Many trout become poor in meat quality after the discharge of sex products when gonad maturation; while fish with less gonad development has higher content of essential amino acids, flavor amino acids in their muscle tissues. Therefore, the implementation of reproductive and developmental regulation of fish is an important guidance for cultivating new lines of cultured fish with excellent traits.
Since the advent of the first batch of transgenic fish (Zhu Z, Li G, He L, et al. Novel gene transfer into the fertilized eggs of goldfish (Carassius auratus L. 1758). Z angew Ichthyol, 1985, 1:31-34.), with the purpose of providing sources of high-quality food proteins, more than 30 kinds of transgenic fish have been successfully developed. Transgenic fish having been genetically modified has excellent traits as fast growth, high feed conversion efficiency, freeze and cold resistance, and strong disease resistance and the like. However, so far there is not a single case of industrial farming of transgenic fish, the bottleneck factor that restricts the industrialization of transgenic fish is the concern on its potential ecological risk. The potential ecological risk of transgenic fish is substantially closely related to its reproductive characteristics from a scientific view. The control of transgenic fish reproduction can fundamentally solve the potential ecological risk of transgenic fish (Hu Wei, Wang Yaping & Zhu Zuoyan. Progress in the evaluation of transgenic fish for possi-ble ecological risk and its containment strategies. Science in China Ser C-Life Sci. 2007, 50:573-579.).
GnRH antisense transgenic strategy is currently the preferred technique to control the reproduction of transgenic fish (Maclean N, Laight R J. Transgenic fish: an evaluation of benefits and risks. Fish and Fisheries, 2000, 1:146-172). The technique firstly represses the expression of GnRH gene which is related to fish gonadal development or sexual maturation by antisense transgene such that its gonadal development is inhibited, and reproduction of transgenic fish is controlled; on this basis, the technique restores the physiological fertility of unfertilized transgenic fish, and finally obtains fertility controllable transgenic fish without ecological risk.
However, even if a transgenic fish with complete infertile gonads is obtained by the use of said technique, it is in logical view an incomprehensible paradox how to transfer the “infertility” trait of the transgenic fish to the offspring and establish a line and is difficult to achieve in genetically view. Although it is theoretically possible to recover the physiological fertility of sterile transgenic fish through the artificial supply of exogenous hormones, it is found by relevant studies that the expression of GnRH gene of fish in the fish brain can start as early as the first day of fertilization and development, the laboratory to which the present inventors belonged has found that a supplement of exogenous GnRH gene at this stage will have an inhibition role in the embryonic development and the growth of juvenile fish, these restrictions make the success rate for fertility restoration very low. Moreover, even few sterile transgenic fish has its physiological fertility restored, the amount of sperms or eggs produced are very small, which is unable to meet the needs of large-scale production.
Gal4/UAS system has the potential to become an ideal inducible gene expression system used in the regulation of fish reproduction and development. Galactose regulated upstream promoter element Gal4 is a yeast transcriptional activator similar to the prokaryotic lactose operon, Gal4 protein has both DNA-binding domain and transcriptional activating domain. Upstream activat sequence UAS is another sequence in yeast that is similar to the enhancer sequence in higher eukaryotes. Gal4 activates the transcription of the downstream gene connected to UAS only when UAS is recognized, and has a strong specificity and controllability for the inducible expression of genes (Marnie E. Halpern, Jerry Rhee, Mary G. Goll, Courtney M. Akitake, Michael Parsons, and Steven D. Leach. Gal4/UAS Transgenic Tools and Their Application to Zebrafish. Zebrafish, 2008, 5 (2):97-110.).
Primordial germ cells are the precursor cells of germ cells, dead end (dnd) gene has an important role in the survival and migration of primordial germ cells (Gilbert Weidinger et al. dead end, a Novel Vertebrate Germ Plasm Component, Is Required for Zebrafish Primordial Germ Cell Migration and Survival, Current biology, 2003, (13):1429-1434.), the inhibition on the expression of dead end (dnd) gene can inhibit the normal migration of primordial germ cells, thereby inhibiting the formation and maturation of germ cells.
Zebrafish (Danio rerio) is currently a model fish that is widely used in the study of developmental biology, aquatic biotechnology and the like (Westerfield M. 1993. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). University of Oregon Press, Eugene, Oreg.). It has guiding significance for the establishment of fish on-off reproduction technology, the breed of excellent farmed varieties of economic gonadal abortive fish and the control of exotic fish populations in the natural environment, etc. by taking zebrafish as a model to explore new methods for controlling fish reproduction.